The present invention relates to techniques for fabricating fluid microcapillaries and optical waveguides in a single device using the same fabrication process, and to optical/fluid flow systems for miniaturized optical excitation and detection devices for use in optical sampling of small volumes of fluid.
Fluorescence is one of the main tools biologists and biochemists have used to understand the processes of life. It is routinely used in laboratories to monitor the progress of biochemical reactions in vitro or in vivo, to measure the produced, consumed, and available quantity of important biological agents, such as proteins or DNA, and to study the dynamics of living cells at a molecular scale. Fluorescence has several advantages as a probe for biochemical processes. Extremely small amounts of dye can yield a large signal, minimizing the perturbation of the natural process. Some dyes exhibit drastic changes in fluorescence quantum yield or in emission spectrum on binding, giving a high specificity of the signal on the environment. Furthermore, it is more available and practical than radioactive markers. Finally, improvements in detection apparatus have shown that fluorescence can reach the highest sensitivity possible; namely, temporally resolved single molecule detection.
The optics available for use in the detection of low level light emissions, such as fluorescence, phosphorescence, chemoluminescence, Raman scattering, etc., which will hereafter be referred to generally as fluorescence, the use of lasers as light sources, and detection methods like confocal microscopy and two-photon excitation have pushed detector probe volumes to lower and lower sizes. Such small probe volumes have provided a higher sensitivity for the detectors, due the decrease in the Raman background from the solvent. These detectors have also brought a wealth of new information on the dynamics of measurement systems through the study of the signal correlation functions. In parallel to that evolution, the amount of re-agent that biochemists have used for their experiments has been decreasing steadily. Smaller and smaller liquid handling systems are now being fabricated to handle the new needs.
Ultimately, processes and methods from the semiconductor industry are used to create the evolved structures necessary to control the small amounts of fluid. However, the techniques used for the detection process have not followed the same path. Since efficient single-fluorophore detection and analysis could lead to significant new applications in analytical chemistry and biology, improved devices and methods of fabricating such devices are needed.
It is an object of the present invention to provide an improved apparatus for efficiently collecting light emitted from femtoliter volumes of fluids.
It is another object of the invention to provide a device for delivering laser radiation to femtoliter volumes of fluid in order to efficiently excite light emission, and to provide a unique method for fabricating such a device.
It is another object of the invention to provide a new integrated device for performing optical detection and spectroscopy of a single light-emitting molecule in a highly confined, flowing fluid and, more particularly, to detect target materials such as single molecules or single cells in a very small volume.
It is another object of the invention to provide a device for delivering laser radiation to a small volume of fluid in order to efficiently excite light emission from target materials, by integrating a fluid flow channel with an optical waveguide and by providing for light collection optics for such a device.
It is still another object of the invention to create a significant new apparatus for rapid, continuous flow sequencing of single DNA fragments by efficiently identifying fluorescently labeled bases in a flowing liquid.
A still further object of the invention is to provide a method for fabricating a unitary microcapillary and optical waveguide device for use in single molecule detection.
Briefly, in accordance with the present invention, an integrated fluid channel and optical waveguide is fabricated by simultaneously microfabricating one or more microchannels for the flow of fluids and one or more optical waveguides for providing illumination of such channels. The fabrication process provides miniaturization, integration and parallelism of optical excitation and detection devices for the sampling of various small volumes of fluids by providing at least one submicrometer channel; i.e., a fluid channel preferably having a width smaller than one micrometer and a depth of between about 0.125 xcexcm and about 1.0 xcexcm, depending on the material being monitored, and at least one intersecting optical waveguide. In one embodiment, both the microchannel and the waveguide are fabricated simultaneously on a common substrate using, for example, photolithography and reactive ion etching in dielectric layers on glass or on silicon substrates. The submicrometer fluid channel will be referred to herein as a microchannel or microcapillary. In use, target materials such as dye-labeled molecules may be electrophoretically driven through the microchannel while laser light is coupled into the waveguide which intersects it, to excite the dye and to produce emitted light. An optical detector permits observation of the light emitted from individual molecules.
The dimensions of the microchannel and the waveguide provide a small, well-defined interaction volume in the channel, resulting in a lower probability of having more than one target element such as a molecule in the interaction volume and providing decreased background noise. The small portion of the microchannel structure subjected to the excitation light reduces background light and increases the efficiency of the detection process. Although the detection of single fluorophore molecules is a primary objective, similar structures can also be used to study other materials, such as polymer strands, in restricted channels, leading to a better understanding of the electrophoresis phenomenon, for example.
The present invention provides several new devices and techniques for improving the optics and fluid flow control in a monitoring or inspection device, and these are particularly useful, for example, in improving the delivery of laser excitation to fluorescently labeled molecules such as DNA nucleotides. The optical waveguide which is integrated into the same structure as the microchannel structure accurately and reliably aligns the laser with the microchannel, and limits the laser excitation to a very small interaction volume, for the waveguide confines the laser beam to a small, well-defined, propagation path. The structure is capable of providing extremely small interaction volumes, in the sub-femtoliter range, and thus is suitable for the detection of extremely small quantities of fluorophores. Beam confinement in the waveguide is similar to confinement in silica optical fibers, although in the disclosed embodiment, a rectangular waveguide is used. Although the invention is described herein with a single fluid microchannel intersecting a single waveguide, it will be understood that, if desired, multiple, parallel, spaced microchannels may be provided, with a single waveguide intersecting all of them, so that light passes through the respective interaction regions in sequence. In addition, multiple parallel waveguides may be provided to intersect a single microchannel or to intersect multiple parallel microchannels.
In one embodiment of the invention, one or more microchannels are fabricated on glass or silicon substrates with minimum channel widths of about 1.0 micrometers for glass or 0.6 micrometers for silicon. Glass-based substrates may include a variety of materials, such as Pyrex, Corning Borofloat, UV grade fused silica, and microscope coverslips. In each case, the substrates are thoroughly cleaned as a first step, and are then covered with a 1.0 micrometer thick layer of doped silicon oxide, preferably deposited by PECVD. This yields a layer of nonstoichiometric silicon oxide, with an index of refraction of around 1.51, which forms a dielectric layer in which one or more microchannels and one or more integral waveguide structures are to be fabricated. The resulting wafers, or samples, are then covered with an opaque layer, such as an aluminum film, which is to be used as an etch mask. At least one waveguide and one or more microchannels of desired shape and width are then exposed in a resist layer, and the resist pattern is transferred to the mask layer in conventional manner. The dioxide layer is then dry etched through the mask pattern in a CF4/O2 plasma to transfer the microchannel and waveguide patterns, and the mask layer is then removed.
Glass-based microchannels are covered with a microscope coverslip coated, for example, by spinning, with a thin layer of poly-dimethylsiloxane (PDMS) polymer. Since PDMS is hydrophobic in its natural state, a short oxygen plasma treatment is used to modify this characteristic. The coverslip is then placed in contact with the patterned dioxide layer on the substrate, and bonding spontaneously occurs.
The waveguide for illuminating the microchannel is fabricated using the same photolithography and etching steps that are used for fabricating the microchannel. Channels are etched in the one micrometer thick layer of nonstoichiometric silicon oxide on each side of the waveguide to form a ridge which is used as the waveguide. To compensate for the presence of defects in the oxide layer, a beam-coupling region of the waveguide, which receives illuminating light from a suitable source, is fabricated to have a width of 100 micrometers. However, the waveguide preferably narrows down as it approaches the microchannel, with the rate of narrowing being slow enough to convert the energy of the laser light from the fundamental mode of the wide waveguide to the fundamental mode of the narrow waveguide. The laser light may be coupled directly into the wide end of the ridge waveguide, or in the alternative, a grating may be fabricated lithographically at the large end of the waveguide to facilitate coupling. The grating may be produced by covering the sample with a resist layer and exposing the resist through a pattern using e-beam lithography. The resist is developed and 100 nanometers of the silicon oxide dielectric forming the ridge are etched through the pattern, using conventional reactive ion etching (RIE).
The foregoing fabrication procedures permit simultaneous formation of submicron fluid channels and automatically aligned ridge waveguides in a single integral photolithographic and etching process. The process allows the waveguide to have a cross sectional area as small as one micrometer at the region where it approaches the microchannel, thereby providing a femtoliter interaction region in the corresponding fluid channel. For single molecule detection, as well as for the detection of small numbers of molecules, signal-to-noise ratios are vastly improved, since the effective density of fluorophores in the fluid channel is vastly increased.
An alternative, and preferred, process for fabricating an integral microchannel and optical waveguide device includes formation of a sacrificial layer which is shaped to define the desired microchannel configuration on a substrate such as fused quartz. A high refractive index layer of a material such as doped silicon dioxide is then deposited on the top surface of the substrate, covering the shaped sacrificial layer. Conventional lithographic and etching processes are then used to fabricate a ridge waveguide by etching channels in the silicon dioxide layer, and to etch irrigation holes through the silicon dioxide layer to expose the sacrificial layer. A grating may then be etched on the top surface of the ridge waveguide, as previously described, and the sacrificial layer is removed, using wet chemistry supplied through the irrigation holes. Thereafter, the irrigation holes are covered to close the fluid channel and, if desired, the channels defining the ridge waveguide may be filled by depositing a material having a low index of refraction. Thereafter, access wells may be etched through the covering material to provide access to the microchannel.
The fabrication of integral microchannel and waveguide structures utilizing the foregoing microlithographic techniques on the same substrate to form an integrated unit provides the foregoing structures at a low cost. Furthermore, multiple microchannel systems fed by one or more cross-connecting, single-mode waveguides can be readily fabricated.
Large angle collection optics can be integrated into the microcapillary-waveguide structure of the present invention, thereby enabling geometrical collection efficiencies of up to 80%. Furthermore, since the laser beam path is completely separated from the detection system optics, laser scattering from optical surfaces in the detection system is avoided. Because the present device reduces scattering noise and increases collection efficiency, a continuous wave excitation laser can be employed, and such a laser is less complex and less expensive than a pulsed laser/gated detection system.
The present device is extremely versatile, providing multimode optical waveguides and capillaries fabricated with dimensions on the order of tens of microns.